The research proposed is directed toward obtaining information on the location and arrangement of specific DNA sequences and chromatin-associated proteins and their interactions in euchromatin and heterochromatin. The studies involve a combination of ultrastructural and biochemical analyses of the material. Using appropriate tags, RNA polymerase III and histone H1 will be located by immunological staining of chromosomes and chromatin at the light and electron microscope levels, respectively. We will attempt to optimize antigen-antibody reactivity and to preserve ultrastructural features of chromatin fibers in order to permit routine quantitative studies on chromosome-associated proteins by immunoelectron microscopy by microinjecting antibodies into living cultured cells via fusion with red blood cells which are loaded with IgGs during hypotonic hemolysis followed by lysis under conditions which optimize chromatin dispersal. If successful, this protocol will be utilized to localize enzymes and other DNA-binding proteins along chromatin strands. We also are attempting to perform in situ hybridization to preparations adsorbed to electron microscope grids in order to do fine structure mapping of DNA sequences in whole mount metaphase chromosomes and in order to identify specific active genes by virtue of hybridization to nascent RNA chains. Features of constitutive heterochromatin, typified by the centromere region of mouse metaphase chromosomes, will be investigated by two-dimensional fractionation of restricted centromere-localized DNA sequences, their mapping within the centromere by in situ hybridization at the electron microscope level, cloning of interesting sequences, elucidation of centromere-specific polypeptides and a search for DNA sequence specific-protein interactions, especially those involved in kinetochore placement at the centromere region. These data will be compared with similar studies on the facultative heterochromatin which comprises the inactive X chromosome of mammalian cells. Human cells containing 3-4 Barr bodies per interphase nucleus will be analyzed for X-specific DNA sequences by restriction and two-dimensional separation and for X-specific proteins by sequences by restriction and two-dimensional separation and for X-specific proteins by comparative two-dimensional gel analysis. These studies will be analyzed in light of models for X inactivation.